The invention relates to combined optical and electrochemical detection, and more particularly, to an apparatus and method for use in the performance of such studies.
Without limiting the scope of the invention, its background is described in connection with a combined electrochemical and optical apparatus for luminescence imaging of microenvironments and methods that may be used in the performance of such imaging.
Fluorescence imaging is one of the most valuable methods for analyzing microenvironments, particularly cellular microenvironments, and can be expected to find broader applicability as the rapidly growing computer and video industries provide new tools/hardware for fluorescence imaging. Although promising in theory, fluorescence microscopy is limited by the number of analytes that are amenable to fluorescent detection schemes. While it is straightforward to image analytes with native fluorescence, analytes labeled with a fluorophore, or analytes that can interact with a fluorescent indicator (e.g., H+, Ca2+, O2), imaging other species such as electroactive analytes is problematic. For example, a major intracellular electroactive analyte of particular importance in understanding cellular metabolism, hydrogen peroxide (H2O2) is presently unamenable to continuous fluorescent analysis on a cellular resolution level.
The motivation for imaging hydrogen peroxide and other reactive oxygen species (ROS) in biological cells and tissue stems from their role in oxidative stress and oxidative burst events. Unfortunately, the in situ monitoring of ROS dynamics on the cellular level is limited by existing technology. Currently, the most common microscopic method for imaging hydrogen peroxide involves loading cells with dichlorofluorescin and quantitating the hydrogen peroxide by following the oxidation of dichlorofluorescin by hydrogen peroxide to produce fluorescent dichlorofluorescein. This technique can be used for detecting ROS liberated in or diffusing in the cytosol of the cell but it is not a direct reporter for ROS generated at the plasma membrane and/or released to the exterior of the cell. Hydrogen peroxide fiber-optic sensors or biosensors that are suitable for single cell analysis by virtue of the ability to acquire a continuous real time measurement with (sub)micrometer spatial resolution have not been demonstrated.
Nicotinamide adenine dinucleotide in its reduced form (NADH), is a biologically important coenzyme that is both fluorescent and electroactive. The quantitation of this molecule is of great interest in chemistry, biology, and medicine. For example, in addition to its use as a metabolic activity marker, NADH is an ideal biosensor reagent since it can modulate the activity of over 200 different dehydrogenases [Pantano, P. and Kuhr, W. G. (1995) Electroanalysis 7, 405-416]. Unfortunately, both the NADH fluorescence and electrochemical measurements are difficult to perform. For the NADH fluorescence measurement, in addition to its low quantum yield, there is significant biological (auto)fluorescence in the same spectral region as the NADH emission. Furthermore, the throughput of borosilicate glass and silica-based optical fibers is attenuated greatly in the ultraviolet spectral region where the NADH excitation wavelength lies (i.e., 340 nm). Finally, improving a CCD camera""s 400-500 nm quantum efficiency (the NADH emission spectral region) by employing back-thinned chips is prohibitively expensive. Also, existing electrochemical fiber-optic NADH-biosensors lack the resolution required for imaging purposes.
What is needed is an apparatus and system that readily permits concurrent luminescence imaging and electrochemical sensing of important analytes with a microscopic level of resolution.
The present invention is directed to apparatus and methodology for concurrent fluorescence imaging and chemical sensing of an electroactive analyte through a fiber optic electrode with resolution on the microscopic level.
The fabrication and characterization of imaging fiber electrodes (IFEs) is presented, and the use of an electrochemically-modulated, fluorescence-based, imaging-fiber electrode chemical sensor (IFECS) is demonstrated.
In one embodiment, the invention provides a fiber optic electrochemical sensor for detecting an analyte including a fiber optic layer, a electrically conductive translucent metallic layer, and a light energy absorbing dye layer. The fiber optic layer of the fiber optic electrochemical sensor may be a fiber optic bundle that includes one or more of individual optic fibers, wherein each individual optic fiber has a diameter of less than 20 micrometers. In one embodiment, the fiber optic electrochemical sensor includes a fiber optic bundle with individual optic fibers, wherein each individual optic fiber has a diameter of less than 10 micrometers and wherein the bundle has a diameter of less than 2 millimeters. The sensor also has an electrically conductive translucent metallic layer, and a light energy absorbing dye layer wherein the fiber optic electrochemical sensor is capable of electrochemical regeneration. Due to its microscopic resolution capabilities, the apparatus and method of the present invention is particularly applicable to studies of cells and tissues, however, the scope of the invention is not limited to biological microenvironments but rather is applicable to any microenvironment in which fine spatial resolution of imaging concurrent with chemical sensing is needed.
In alternate embodiments, the electrically conductive translucent metallic layer of the fiber optic electrochemical sensor is between 10 and 100 nm thick. The metallic layer may be of any electrically conductive metal or metal oxide that may be applied in a translucent layer wherein xe2x80x9ctranslucentxe2x80x9d, used interchangably herein with xe2x80x9ctransparentxe2x80x9d, means susceptible to the through-passage of light energy. In examples provided herein, the fiber optic electrochemical sensor electrically conductive translucent metallic layer is a sputter-coated 20-23 nm layer of gold.
In alternate embodiments, the light energy absorbing dye layer of the fiber optic electrochemical sensor is selected from the group consisting of, e.g. fluorochromes, fluorescent enzyme conjugates, fluorescent substrates and chromophores. In one example, the analyte to be detected is a cellular reactive oxygen species and the light energy absorbing dye layer includes a rhodamine dye.
In another example the analyte to be detected is cellular NADH and the light energy absorbing dye layer includes a ruthenium containing luminophore.
In another embodiment of an imaging system according to the present invention, a fluorescence based imaging fiber electrode chemical sensor system includes a fiber optic electrochemical sensor, a potentiomer or equivalent means for measuring ion flux, a microscope including a light source and an objective lens. The objective lens communicates light from the source to the fiber optic electrochemical sensor and receives light returning from the sensor and provides a means for recording light returning from the sensor though the objective. Potentially useful recording devices include CCD cameras, linear arrays and xy active matrix detectors.
The invention provides in one embodiment a method for preparing a imaging fiber electrode including the steps of; polishing a face of the fiber optic bundle, silanizing the face using a mercapto-trimethyoxysilane, and sputter coating the silanized face to deposit a 10-30 nm thick semi-transparent metal layer.
In another embodiment, a method for preparing a imaging fiber chemical sensor is provided including the steps of; obtaining a fiber optic electrode having a 15-30 nm gold film on a distal end and an electrically conductive aspect leading from the distal end through a lateral dimension of the fiber optic electrode, coating the fiber optic electrode with an ion-exchange polymer and applying a luminescent reporter group.
In one example of an ion-exchange polymer according to the present invention poly(tetrafluorethylene)polymers having characteristics of NAFION polymers have been found suitable.
In one embodiment, the invention provides a method of constructing and using electrochemically-modulated, fluorescence-based, imaging-fiber electrode chemical sensors (IFECS). An imaging fiber distal tip is coating with a semi-transparent metal layer to create an IFE. In an alternate embodiment the imaging fiber is sputter coated with a semi-transparent layer of gold although alternate coating methods with alternate metallic compounds and metal oxides may be applicable.
By xe2x80x9csemi-transparentxe2x80x9d or xe2x80x9ctranslucentxe2x80x9d according to the present invention, is meant a layer of gold sufficiently thin for light to be transmitted through it and yet thick enough to serve as an electrode. The term xe2x80x9cgoldxe2x80x9d as used in the present invention includes gold and any alloy containing gold. A range of metal thickness of 10 to 100 nm may be expected to perform these dual roles under appropriate circumstances. A light energy absorbing dye such as a fluorescent redox dye is immobilized across the IFE face to create an IFECS. Alternate light energy absorbing dyes are known in the art and many are commercially available. Potentially applicable analyte responsive or reporter dyes may include either, or a mixture of, light emitting and light absorbing dyes or may include other materials such as enzymes, or antibodies, or chemical compounds. Potentially applicable luminescent reporters in addition to the specific examples of RBITC, NADH and tris(2,2xe2x80x2-bipyridyl)ruthenium provided herein may include for example such fluorochromes as: nile blue A, rhodamine 123, rubrene, rhodanile blue, eosin, TRITC-amine, quinine, fluorescein W, acridine yellow, lissamine rhodamine B sulfonyl chloride, erythroscein, Texas Red, phycoerythrin, flavin adenine dinucleotide (FAD), carboxy seminaphthorhodafluor, and naphthofluorescein. Fluorescent enzyme conjugates may be employed in some circumstances in addition to substrates such as for example: fluorescein mono-B-D-galacto-pyranoside, resorufin, B-d-glucuronide, 8-acetoxypyrene 1,3,6-trisulfonic acid trisodium salt, Coenzyme A (1-pyrene butanoic acid)ester, Fluo-3, and Quin-2. Potentially applicable chromophores include for some examples not intended to be limiting: iron-salicylate complex, Indamine dye, Hopkins-Cole dye, quinone-imine dye, Fe(SCN)+2, Malachite Green, cresol red, diphenylcarbazone disulphonic acid, chrome bordeaux B, calmagite and ninhydrin dye. Applicable analyte responsive dyes may include either, or a mixture of, light emitting and light absorbing dyes or may include other materials such as enzymes, or antibodies, or chemical compounds. The reporter dyes may further include pairs of donor and acceptor molecules such as those known in the art to participate in fluorescence resonance energy transfer (FRET) in which excitation between two dye molecules is transferred from a donor molecule to an acceptor molecule without emission of a photon. Known examples of FRET pairs include Fluorescein/Tetramethylrhodamine, IAEDANS/Fluorescein, EDANS/DABCYL, Fluorescein/Fluorescein, BODIPY FL/BODIPY FL and Fluorescein/QSY-7 dye.
An electroactive analyte is imaged by monitoring the change in the immobilized redox dye""s fluorescence following its homogeneous electron transfer reaction with an electroactive analyte. Fluorescence images were collected through the IFECS and detected by an imaging system such as for one example an epi-fluorescence microscope/CCD imaging system. Instead of using a CCD camera, other sensitive detectors such as for example linear array detectors or XY matrix photodetectors may be included in the imaging system. Cyclic voltammetry and fluorescence microscopy may be utilized to characterize the electrochemical and fluorescence properties of IFECSs.
In one embodiment of the present invention, reversible voltammetry was observed for the redox couple at IFEs permitting the sensor to be recharged and used for multiple measurements. In on example, IFECSs using RBITC as the fluorescent redox dye and NAFION as the immobilization polymer were fabricated to detect hydrogen peroxide. The IFECS""s RBITC-fluorescence was decreased by xcx9c27% upon exposure to 0.25 mM hydrogen peroxide and xcx9c95% of the original fluorescence was observed following the electrochemical regeneration of the NAFION-immobilized RBITC. These IFECSs provide for remote fluorescence imaging of an electroactive analyte, which may be performed through the actual fiber-optic electrode itself and have the capacity to regenerate.
The invention provides a dual system that includes a potentiostat/electrochemistry system together with features of a combined imaging and chemical sensing approach. The combined imaging and chemical sensing (CICS) approach, permits viewing a sample and measuring surface chemical concentrations using an optical imaging fiber [Bronk, K. S., Michael, K. L., Pantano, P., Walt, D. R. (1995) Anal Chem 67:2750; Pantano, P., Walt, D. R. (1995) Anal Chem 67:481A; and Panova, A. A., Pantano, P., Walt, D. R. (1997) Anal Chem 69:1635]. According to the CICS approach to imaging, a high resolution imaging fiber may include thousands of individual optical fibers in a diameter range measured in xcexcm melted and drawn together such that an image can be carried and maintained in a coherent manner from one end to the other. As an example a 350-micrometer-diameter distal fiber surface, containing xcx9c6000 optical sensors of xcx9c3 micrometer diameter, is coated with a uniform, planar sensing layer that can measure chemical concentrations with spatial accuracy, yet is thin enough so that it does not compromise the fiber""s imaging capabilities. By combining the distinct optical pathways of the imaging fiber with the spatial discrimination of a charge coupled device (CCD) camera, visual and fluorescence measurements can be obtained with 4-micrometer spatial resolution over thousands of square micrometers using a CICS approach. In the present invention, the concept of imaging fiber electrodes (IFE) is united with the CICS approach. The present inventors have developed a novel thin film fiber optic electrode sensor array and apparatus that permits both electrochemical and luminescence imaging through the actual sensor itself.
In one embodiment, the present invention provides a new apparatus and method for remote hydrogen peroxide imaging which unites the fabrication of imaging fiber electrodes with the combined imaging and chemical sensing approach. The present combined devices are termed herein xe2x80x9cimaging fiber electrode chemical sensors (IFECSs).xe2x80x9d In brief, an imaging fiber""s distal tip is metalized to serve as an electrode (i.e., an IFE) before a light energy absorbing dye such as a fluorescent redox dye is immobilized across the IFE""s distal face. Finally, in the case of a fluorescent redox dye, the redox state of the bound dye (and its initial fluorescence) is regenerated by application of a suitable potential across the IFE surface. While fluorescence and electrochemistry (i.e., electrodes and optical fibers) have been united previously, the present IFECSs permit remote fluorescence imaging to be performed through the actual fiber-optic electrode itself.
In one embodiment of the present invention there is provided a new technique for remote NADH imaging wherein combined imaging and chemical sensing of Ru(bpy)32+-Electrogenerated Chemiluminescence is achieved. This has been accomplished by fabricating imaging fiber electrode (IFE) sensors. In one example, an imaging fiber""s distal tip is metalized to serve as an electrode (i.e., an IFE). The IFE is coated with a thin, planar layer of NAFION doped with Ru(bpy)32+ to produce an IFE Sensor. An electrical contact is made to the metal layer such that Ru(bpy)32+ can be oxidized to Ru(bpy)33+ and electrochemically regenerated following the application of an appropriate potential. Following the diffusion of NADH into the IFE sensing layer, the Electrogenerated Chemiluminescence emission is captured and analyte concentrations quantitated using a CCD-based imaging system.